title: Rapid steps in the glmS ribozyme catalytic pathway: cation and ligand requirements.
abstract: The glmS ribozyme is a conserved riboswitch found in numerous Gram-positive bacteria and responds to the cellular concentrations of glucosamine 6-phosphate (GlcN6P). GlcN6P binding promotes site-specific self-cleavage in the 5' UTR of the glmS mRNA, resulting in downregulation of gene expression. The glmS ribozyme has previously been shown to lack strong cation specificity when the rate-limiting folding step of the cleavage reaction pathway is measured. This does not provide data regarding cation and ligand specificities of the glmS ribozyme during the rapid ligand binding chemical catalysis events. Prefolding of the ribozyme in Mg(2+)-containing buffers effectively isolates the rapid ligand binding and catalytic events (k(obs) > 60 min(-1)) from rate-limiting folding (k(obs) < 4 min(-1)). Here we employ this experimental design to assay the cations and ligand requirements for rapid ligand binding and catalysis. We show that molar concentrations of monovalent cations are also capable of inducing the formation of the native GlcN6P binding structure but are unable to promote ligand binding and catalysis rates of >4 min(-1). Our data show that the sole obligatory role for divalent cations, for which there is crystallographic evidence, is coordination of the phosphate moiety of GlcN6P in the ligand-binding pocket. In further support of this hypothesis, our data show that a nonphosphorylated analogue of GlcN6P, glucosamine, is unable to promote rapid ligand binding and catalysis in the presence of divalent cations. Folding of the ribozyme is, therefore, relatively independent of cation identity, but the rapid initiation of catalysis upon the addition of ligand is stricter.
authors.affiliation: Department of Microbiology and Molecular Genetics, The Markey Center for Molecular Genetics, Stafford Hall, 95 Carrigan Drive, University of Vermont, Burlington, Vermont 05401, United States.
authors.forename: Krista M
authors.initials: KM
authors.lastname: Brooks
authors.forename: Ken J
authors.initials: KJ
authors.lastname: Hampel
mesh_terms: D001412:Bacillus subtilis / Q000378:metabolism*; D001665:Binding Sites; D055162:Biocatalysis; D005944:Glucosamine / Q000031:analogs & derivatives / Q000378:metabolism; D019298:Glucose-6-Phosphate / Q000031:analogs & derivatives / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D008274:Magnesium / Q000737:chemistry*; D009690:Nucleic Acid Conformation; D009994:Osmolar Concentration; D012329:RNA, Bacterial / Q000737:chemistry / Q000378:metabolism*; D016337:RNA, Catalytic / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D058928:Riboswitch
chemical_list: D008024:Ligands; D012329:RNA, Bacterial; D016337:RNA, Catalytic; D058928:Riboswitch; C001293:glucosamine 6-phosphate; D019298:Glucose-6-Phosphate; D008274:Magnesium; D005944:Glucosamine